For this reason, it is impossible to estimate visually that the average precursor only underwent two divisions in these distributions.Īll statistics except “Fraction Diluted” require modeling to estimate the fraction of cells in each generation. Very few cells in the starting population undergo more than four divisions (insets), but these rare cells are highly disproportionately represented in the CFSE distribution. Notice how the CFSE distribution (distribution of divided cells) looks completely different than the distribution of precursor cells. The inset shows the fraction of cells that undergo one to six divisions (in each case, 70% undergo no divisions) the percentage of cells that undergo only one division is shown above the right most bar. The distribution of the number of divisions for cells in the starting population is shown in each inset the variance on this distribution (SD D) increases from ☑ (top) to ☒ (bottom). ( B) Hypothetical CFSE distributions for cell cultures, for which PF is 30% and PI is 2. Grey numbers indicate the generation number. ( A) Shown are hypothetical CFSE distributions for cell cultures, in which the precursor frequency (PF) ranges from 20% (top) to 2.5% (bottom), and the average number of divisions that a responding cell has undergone (PI) increases from 3 (top) to 6 (bottom), as might happen with longer culture times. 1 All figures and analyses in this article are based on hypothetical data generated for illustrative purposes.Įffect of culture time, precursor frequency, and division variance on proliferation distributions. Figure 1 and Table 2 show values for these statistics for hypothetical CFSE distributions. Thus, authors should be careful to clarify which statistic they are reporting.
#Flowjo table editor software#
Table 1 lists a number of easily-calculable statistics to characterize CFSE distributions some confusion arises from the same name having been given to distinct statistics by various software packages. Statistical Analysis of CFSE Distributions Second, the ability to resolve generations depends on the CV of each fluorescent peak (CV F) often, as divisions increase, the CV F may increase, leading to a “smearing” of the populations in higher generations. First, there is a limit to how brightly-stained the original population can be seven divisions results in cells that have 1/128th (<1%) of this fluorescence, which may not be much above autofluorescence. In general, the limit on the number of resolvable generations is about 8 this is governed by two limitations. those that have divided a specified number of times. When good staining techniques are used, the CFSE assay can be used to separately quantify the number of cells in the culture that have not divided vs. The introduction of the CFSE assay provided researches with the ability to derive those values, and, with sophisticated mathematical models, estimate values such as death rates, change in proliferation rates over time, and more ( 5- 7). Both of these values have biological relevance. Neither assay could be used to determine the precursor frequency (i.e., what fraction of original cells ever underwent division) nor the distribution of the number of divisions that responding cells underwent.
Cell counting provides an assessment of the total expansion of the culture (i.e., how many cells are present at termination compared to initiation) 3H-T provides an assessment of how many cells were in cell cycle during the pulse phase (usually the last few hours prior to termination). Prior to the introduction of this assay, there were two dominant methods for quantifying proliferation: cell counting and 3H-thymidine ( 3H-T) uptake. The basic principle is well-described, and relies on the two-fold dilution of fluorescence accompanying each cellular division as the fluorescently-tagged cellular proteins are allocated equally to each daughter cell. A flow cytometric assay based on the dilution of carboxy fluorescein diacetate succinimidyl ester (CFSE, also known as CFDA-SE) fluorescence during culture has become the standard for this purpose ( 1) when done well, analysts can enumerate cells that have divided as well as quantifying how many divisions each cell has undergone ( 2- 4).
An important assessment of cellular function, particularly in immunology, is the proliferative capacity of cells under different conditions.
0 kommentar(er)
